An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors

ABSTRACT Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10−/− mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridium cluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.


Introduction
Inflammatory bowel disease (IBD) is a chronic inflammatory state of the gastrointestinal tract, including Crohn's disease (CD) and Ulcerative colitis (UC). The etiology of IBD is unknown, but the collective evidence indicates that a dysregulated immune response to commensal enteric microorganisms (one of the hallmarks of IBD) leads to uncontrolled chronic inflammation in the genetically susceptible host. 1,2 Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used for various inflammatory conditions, including IBD-associated extraintestinal manifestations. However, mucosal injury and ulcers are common adverse effects of NSAID use and are therefore believed to exacerbate inflammation in patients with IBD. 3 However, the current literature on the association between NSAIDs and IBD is inconclusive. 4,5 Evidence to date suggests that the gut microbiota might play a critical role in NSAIDpromoted inflammation. 6 For example, germ-free rats are resistant to NSAID-induced intestinal damage 7 and an increase in the relative-abundance of potentially harming bacteria such as Rikenellaceae, Pseudomonadaceae, Propionibacteriaceae, and Puniceicoccaceae was observed in individuals taking ibuprofen. 8 Furthermore, an increased expansion of Gram negative taxa such as Escherichia/Shigella, Bilophila, and Bacteroides was reported in the ileum of rats with indomethacin-induced enteropathy. 9 In addition, consumption of a Bifidobacterium strain reduced NSAID-induced ulcerations in healthy volunteers. 10 No reports on how NSAIDs might affect the microbiota of patients with IBD or how the presence of certain IBD-associated bacteria may affect the response of the patients when treated with NSAIDs have been found in the literature.
Genetic background and environmental factors can contribute to disease triggered by pathogenic commensals, so-called pathobionts. 11 Adherentinvasive Escherichia coli (AIEC) is a gut pathobiont widely prevalent in IBD patients in Western countries, up to 10% and 62% in patients with UC and CD, respectively. 12,13 AIEC has been reported to trigger inflammation in genetically predisposed mice and even promote fibrosis in chemically induced colitis. 14 A recent study reported that AIEC might be associated with the early phase of recurrence in patients with CD, 15 further indicating their potential contribution to CD pathogenesis.
NOD-like receptors (NLRs) are an important family of pathogen recognition receptors (PRRs) that sense pathogen and commensal associated molecular patterns (PAMPs), or danger associated molecular patterns (DAMPs), leading to coordinated innate and adaptive immune responses. One of the most intensely studied members of this NLR family is NLRP3, whose activation leads to the formation of the NLRP3 multiprotein inflammasome complex. Formation of this complex activates caspase-1 and subsequent cleavage of downstream substrates such as the effector inflammatory cytokines, pro-IL1β and pro-IL18. 16 Activation of NLRP3 has been reported in pre-clinical models of intestinal infection and colitis, in human IBD tissue and by AIEC in macrophages. 17,18 Genomewide association studies have identified an SNP in the region of the NLRP3 gene possibly contributing to disease susceptibility in CD, and a recent study reported a loss-of-function mutation in the CARD8 domain, causing NLRP3 activation and CD. 19 NLRP3 has also been shown to play a crucial role in NSAID-induced enteropathy. 20 One of the hallmarks of chronic inflammation is the increased death (apoptosis) of intestinal epithelial cells in patients with IBD. 21 One initiator caspase, which acts as a central regulator of the crosstalk and plasticity between multiple cell death pathways (apoptosis, necroptosis, and pyroptosis) and inflammation, is Caspase-8. 22 Various studies have shown the role of Caspase-8 in intestinal inflammation. 23,24 Activation of death receptors (e.g., TNFR), Toll-like receptors (TLR3/-4), the z-form nucleic acid sensor (ZBP1), or the intracellular RNA sensor RIG-I leads to the recruitment of Caspase-8 to the intracellular signaling complex, followed by its autoproteolytic cleavage and activation. Active caspase-8 cleaves its substrate caspase-3/7, initiating apoptosis. In addition, active caspase-3/7 can also cleave poly(ADPribose) polymerase (PARP), resulting in aberrant DNA repair and apoptosis. 22,25 Active Caspase-8 inhibits necroptosis by suppressing the function of receptor-interacting protein kinase 1 (RIPK1) and RIPK3, to inhibit the hosphorylation of the poreforming mixed lineage kinase domain-like (MLKL) protein. 2 Another cell death mechanism provoked by inflammation and microbial infections, e.g., Salmonella, is pyroptosis. This cell death can be induced by the canonical activation of Caspase-1 or via the non-canonical caspases, Caspase-11 (mice) and Caspase-4/5 (humans), 2 leading to the cleavage of its main executor gasdermin D (GSDMD). Interestingly, Caspase-8 has been shown to cleave GSDMD leading to pyroptosis. 26 Caspase-8 can act both upstream and downstream of NLRP3 inflammasome activation. On the one hand, it triggers NLRP3 activation via both its enzymatic activity and scaffolding function. [27][28][29][30] On the other hand, NLRP3 can regulate Caspase-8 activation in epithelial cells independent of Caspase-1 activation or cytokine production. 31 Based on the collected evidence to date, we hypothesized that the apparent inconsistency in reports regarding the impact of NSAIDs on IBD disease activity could be explained by a dysregulated microbiota composition and the presence of pathobionts such as AIEC in these patient cohorts. In this study, we used interleukin-10 deficient (IL10 −/− ) mice, a pre-clinical IBD model used to mimic the genetic predisposition in IBD, to test our hypothesis. We show that AIEC and NSAIDs combine to induce/ exacerbate colitis and cell death via the NLRP3 inflammasome and Caspase-8 with evidence of NLRP3-Caspase-8 cross-talk contributing to this overall phenotype. This provides new insights into how NSAIDs in the presence of an opportunistic gut pathobiont could worsen IBD symptoms.

AIEC-mediated sensitization of IL10 −/mice to piroxicam induced-inflammation is associated with activation of the inflammasome, apoptosis, and pyroptosis
To test our hypothesis, we examined the ability of AIEC-HM605, a strain of AIEC isolated from colonic biopsies of a patient with CD, 32 to sensitize IL10 −/− mice to NSAID (piroxicam) induced inflammation. We developed a model (Figure 1a) whereby initial pre-treatment of IL10 −/− mice with streptomycin facilitated colonization with AIEC 33 ( Figure S1C). Subsequent feeding of these mice with 100 ppm of piroxicam in regular chow for 5 d followed by 9 d of normal chow resulted in colitis ( Figure 1 and Figure S1). The initial colonization of AIEC dropped from 10 9 to 10 4 cfu/g at day 7, followed by a stable AIEC colonization throughout the study ( Figure S1C). A similar degree of AIEC colonization (10 4 cfu/g) was seen in the colon tissue of animals in the AIEC and AIEC/piroxicam groups at day 14 (data not show). Similar changes in disease activity index and body weight loss were observed in the AIEC/ piroxicam group, with a higher non-significant body weight loss at day 14 when compared to AIEC or piroxicam only groups ( Figure S1A-B). In line with other models of colitis, 34 only the mice colonized with AIEC and subsequently treated with NSAID had shorter and heavier colons compared to animals exposed to piroxicam or AIEC alone (Figure 1b). This was accompanied by a loss of colonic epithelial integrity indicated by a significant reduction in expression of colonic Zo1 and Muc2 genes ( Figure 1c) and a significant induction in the expression of inflammatory genes and proteins (Cxcl2, Il17a, Ifng and IFNγ, IL1β, and mKC, Figure 1d-e, S1D-E).
Because the NLRP3 inflammasome has previously been shown to be activated by AIEC strains in vitro and in NSAID enteropathy, 18,20 we analyzed its activation by AIEC/NSAID in our model. Indeed, a significant upregulation of colonic NLRP3, caspase-1, and its downstream target IL-1β was revealed at the gene expression and protein level in the AIEC/piroxicam combination group but not in mice colonized only with AIEC or administered piroxicam alone (Figure 1e-g). We also observed Caspase-8 cleavage, which is indicative of Caspase-8 activation in the AIEC/piroxicam combination group (Figure 1g). To confirm Caspase-8 activation, we analyzed cleavage of its downstream target NEDD4-binding protein 1 (N4BP1). 35 Indeed, cleaved N4BP1 was only detected in AIEC/piroxicam combination group ( Figure S1F). Since Caspase-8 can induce apoptosis in part by cleaving and activating Caspase-3, 36 we analyzed and detected the cleaved form of Caspase-3 in the AIEC/piroxicam group when compared to AIEC or piroxicam only groups (Figure 1g). Consistent with this, cleaved PARP was also detected in the AIEC/piroxicam group (Figure 1g). No activation of necroptosis, which was analyzed by western blotting for phospho-MLKL or RIPK1, was seen in any of the groups (data not shown). When we examined the expression of the main executor and biochemical marker of pyroptosis -cleaved GSDMD -we found it was only expressed in the AIEC/piroxicam group (Figure 1g), indicating a synergistic activation of pyroptosis by AIEC/piroxicam.
Collectively, these observations validate our initial hypothesis indicating that AIEC colonization in a genetically predisposed host renders them susceptible to NSAID-induced inflammation. This inflammation is accompanied by activation of the inflammasome, apoptosis, and pyroptosis.

Colonization with AIEC and piroxicam treatment provoked modest alterations of the gut microbiome in IL10 −/mice
To determine the impact of piroxicam and/or AIEC exposure on the gut microbiome, we conducted 16S rRNA gene analysis on fecal samples collected at day 0 and 14 ( Figure 1a). Across treatment groups, an increase in intra-sample alpha-diversity was observed over time ( Figure S2A), suggesting a gradual recovery of mice microbiomes following Streptomycin treatment. While there is a significant (p < .05) increase in alpha-diversity observed in AIEC, and AIEC/piroxicam treated mice between day 0 and day 14 ( Figure  S2A), this trend was non-significant after falsediscovery rate (fdr) correction.
Visually, the ordination of mice 16S rRNA composition shows that beta-diversity differences are greater in mice colonized only with AIEC and/or treated with piroxicam compared to PBS-treated controls ( Figure S2B). Permutational multivariate analysis of variance (PERMANOVA) tests were performed on inter-sample beta-diversity distances to discern the variables significantly contributing to data dispersion (i.e., variance). This analysis revealed that the days post-piroxicam variable accounted for 14.9% of the 16S rRNA data variance ( Figure S2B).
The compositional bar plots on 16S rRNA data show only taxa with a total aggregated relative abundance across all mice greater than 0.1% (g) Western blot of markers of NLRP3 inflammasome, Caspase-8, apoptosis and pyroptosis markers. n = 3-8/ group for RT-qPCR, protein, western blot, and necroscopy. Data are presented as mean ± SEM. Significance was determined using one-way ANOVA with Bonferroni or Kruskal-Wallis test with Dunn's multiple comparison test, respectively. * p < .05; ** p < .01; *** p < .001.
( Figure S2C). As expected, Escherichia/Shigella are detected at day 0 (i.e., 4 d post-AIEC colonization) of the relevant treated animals. However, significant differences in other individual 16S microbial taxa were not observed between mice over time after fdr correction, although some alterations were observed, e.g., increased abundance in Alistipes and Bacteroides in AIEC/piroxicam group, changes often associated with murine colitis. 37 When individual cecal short chain fatty acids (SCFAs) levels were examined, a general but non-significant increase in propionate and reduction in acetate was observed in the AIEC/piroxicam group ( Figure S1G).
Collectively, the data showed that AIEC and/or piroxicam treatment only had modest effects on gut microbiome composition and SCFAs in contrast to other colitis stimuli. 37

Caspase 8 and NLRP3 inhibitors ameliorate NSAID induced epithelial alterations and inflammation in AIEC colonized mice
To further investigate the role of NLRP3 and Caspase-8 in our model, IL10 −/− mice were administered the selective NLRP3 inhibitor, MCC950, 38  A similar trend in activated T-cells was seen in the mesenteric lymph nodes treated with either of the inhibitors (data not shown). These changes in immune cell populations were accompanied by a reduction in colonic gene expression of acute inflammatory cytokines (Il6, Cxcl2, and Inos); macrophage activation markers (Tnfa, Ccl2) and T-cell markers (Ccl5, Cxcl10, Il17a, and Ifng) ( Figure 3a); all of which were initially altered in the AIEC/piroxicam group (Figure 3a, 1d-e, and S1D-E). Similarly, a reduction in protein levels of IL-6 and IFN-γ was also observed in mice treated with either inhibitor compared to AIEC/piroxicam mice ( Figure 3b). Together, these findings suggest that activation of the NLRP3 inflammasome and Caspase-8 may alter epithelial cell biology and barrier integrity through increased inflammatory cell death exacerbating inflammation which is characterized by the presence of activated T-cells and macrophages in NSAID-exposed IL10 −/− mice precolonized with AIEC.

Differential microbiome-host interactions are associated with effects of NLRP3 and Caspase-8 inhibition
Next, we investigated the effects of the smallmolecule inhibitors targeting NLRP3 (MCC950) and Caspase-8 (Z-IETD-FMK) on bacterial composition in fecal samples collected on day 0 and 12 from IL10 −/− mice with colitis induced by AIEC/ piroxicam ( Figure 2a). No significant changes were detected in 16S rRNA alpha-diversity between the different groups or over time ( Figure S3D). When we investigated the variance associated with sample beta-diversity differences ( Figure S3E), the variance attributable to days post piroxicam was 12.3%, while the treatment grouping variable was associated with an 11.4% variance. The interaction of these two variables was 8.8% (p-value = 0.011).
To avoid over-cluttering of the compositional bar plot of AIEC/piroxicam inflamed mice versus Caspase-8/NLRP3 inhibitor groups, only taxa with a total aggregated relative abundance of 0.2% are shown ( Figure 3c). Visually, the 16S compositions at day 0 showed a higher abundance of Clostridium sensu stricto in the inhibitor treated groups, while at day 12, Clostridium cluster XIVa abundance was seen in mice treated with both inhibitors. However, these did not reach statistically significant after fdr correction, potentially due to the small group size comparisons ( Figure 3c).
Interestingly, the rank order Spearman correlations of microbial taxa versus inflammatory markers showed contrasting results with regard to the inhibitor treatment (Figure 3d). For putative Anaerotruncus, Barnesiella, Blautia, Clostridium XIVa, Oscillibacter, and Robinsoniella, the correlation coefficients were almost exactly opposite in the NLRP3 (MCC950) and Caspase-8 (Z-IETD-FMK) inhibitor-treated groups. These microbial correlation differences occur despite both inhibitors similarly dampening the colonic epithelial and inflammatory gene expression profile (Figure 3a).
When the individual cecal SCFA levels were examined, a non-significant increase in propionate was observed in the Caspase-8 and NLRP3 inhibitor-treated groups compared to the AIEC/piroxicam group ( Figure S3F).
Overall, these data indicate that NLRP3 and Caspase-8 inhibitors target-specific microbiomehost interactions without provoking dramatic alterations on microbial taxa.

Caspase-8 and NLRP3 inhibitors block inflammasome activation, apoptosis, and pyroptosis in AIEC colonized and NSAID exposed mice
AIEC/piroxicam combination in IL10 −/− mice resulted in activation of the inflammasome, apoptosis, and pyroptosis ( Figure 1g). When assaying the expression of genes associated with inflammasome activation and apoptosis in mice administered either the NLRP3 or Caspase-8 inhibitors, no changes were observed at the gene expression levels of Caspase 1 and Caspase 8, while Il1b, Nlpr3, and Caspase 3 were significantly reduced when compared to AIEC/piroxicam group (Figure 4a-b). In support of these findings, western blot analysis showed decreased expression of the inflammatory active cleaved Caspase-1, IL-1β, and IL-18, the pro-apoptotic markers -cleaved Caspase-3 and PARP, and the pyroptotic markers -cleaved Caspase-1, Caspase-11, and GSDMD, in the inhibitor-treated groups compared to AIEC/piroxicam combination group ( Figure 4c). As expected, both the NLRP3 inhibitor (MCC950) and the Caspase-8 inhibitor (Z-IETD-FMK) reduced the expression of their own target proteins, and both inhibitors decreased the expression of the same biochemical markers of inflammasome activation, apoptosis, and pyroptosis ( Figure 4c). Interestingly, western blot analysis indicated that both Caspase-8 and NLRP3 can potentially cross-regulate each other. Caspase-8 inhibition reduced NLRP3 both at the gene and protein levels, while NLRP3 inhibition reduced the expression of active cleaved Caspase-8. Several studies have reported that Caspase-8 may act upstream of NLRP3, by inducing NLRP3 activation via GSDMDmediated K + -efflux. 29 To confirm the involvement of Caspase-8 and its interaction with NLRP3, we assessed the expression of N4BP1, a downstream substrate of Caspase-8. 35 As previously shown in Figure S1F, N4BP1 was cleaved in AIEC/piroxicam combination group, but not in the NLRP3 inhibitor group (Figure 4d), suggesting a role for NLRP3 in Caspase-8 regulation in our model. In line with our expectations, no N4BP1 cleavage was detected in the Caspase-8 inhibitor group (Figure 4d), confirming Caspase-8 inhibition. The downregulation in Nlrp3 gene expression observed in Caspase-8 inhibitor group could be explained by the regulation of NF-κB by Caspase-8.

Discussion
In this study, we investigated if the presence of an IBD-associated pathobiont (AIEC) 13,40 would exacerbate intestinal inflammation and worsen symptoms triggered by subsequent administration of NSAIDs in IL10 −/− mice. If observed, this would support and provide a pre-clinical model and mechanistic explanation for the reported worsening of symptoms by NSAIDs in patients with IBD. AIEC is a gut pathobiont found heterogeneously in patients immunophenotyped by Fluorescence-activated cell sorting (FACS) after gating on CD45 ( Figure S2A-C) and expressed as MFI or percent of specific cell populations. n = 5-6/group. Significance was determined using one-way ANOVA with Bonferroni or Kruskal-Wallis test with Dunn's multiple comparison test, respectively. * p < .05; ** p < .01; ***p < .001. (c) Fecal samples were collected for 16S rRNA analysis at days 0 (upon piroxicam feeding) and 14 (end of trial). Data is presented as with IBD 11 and is associated with the pathogenesis of IBD. Consistent with our hypothesis, we show that AIEC colonization determined the severity of NSAID-triggered inflammation in IL10 −/− mice, which was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, promoting inflammation and cell death.
For this study, we modified a previously reported model of piroxicam-accelerated colitis (PAC) 41 by pre-colonizing IL10 −/− mice with AIEC followed by 5 days of piroxicam exposure. Using this design, we showed that piroxicam can only induce colitis in mice pre-colonized with AIEC. Colitis did not occur in IL10 −/− mice colonized with AIEC alone or treated with the NSAID alone. The inflammation in the AIEC/piroxicam combination group was accompanied by elevated levels of pro-inflammatory cytokines (e.g., IL-6, TNFα, IFNγ) and infiltration of activated immune cells including T-cells and macrophages. Changes in the colonic epithelial structure (altered tight junction proteins, elongated crypts, crypt abscesses, and goblet cell loss) were also observed in these mice. In contrast to the PAC model, 41 the conditions for our model were optimized [i.e., a lower dose of piroxicam (100 vs 200 ppm) and fewer days of feeding (5 vs 9 days) were used] to reduce their adverse effects on animal health after antibiotic treatment and AIEC colonization (data not shown). In the PAC model, ulcerations were detected in severe cases, while these were seldom noted in the AIEC/piroxicam combination group. Alterations in the fecal microbiota in AIEC/piroxicam group were subtle compared to untreated mice and to other pre-clinical models such as the dextran sodium sulfate (DSS)-model. 37 Changes in the experimental design and the less severe impact on epithelial damage in our model compared to, e.g., the DSS-model 34 might have contributed to the subtle microbial alterations.
Previous studies identified that NLRP3 inflammasome-derived IL-1β release plays a critical role in NSAID-induced enteropathy 20 and treatment with NLRP3 inhibitors such as MCC950 ameliorates colitis. 42,43 IL-10 produced from macrophages was also reported to be a negative regulator of NLRP3 when induced by different stimuli. 44 In our study, we found that, in addition to NLRP3 activation, Caspase-8 was also activated in the AIEC/piroxicam combination group. Previous studies have shown that epithelial deletion of Caspase-8 promoted DSSinduced colitis. This effect was associated with increased levels of RIP3 and a reduced number of anti-microbial Paneth cells, alluding to Caspase-8 regulation of necroptosis and inflammation in the pathogenesis of colitis. 24 However, no changes in necroptotic cell death markers such as RIPK1, RIP3, or phospho-MLKL were observed in our study in the control or Caspase-8 inhibitor group.
We found that small-molecule inhibitors targeting NLRP3 and Caspase-8 rescued the inflammatory phenotype observed in the AIEC/piroxicam group, indicating an indispensable role of Caspase-8 and NLRP3 in AIEC/NSAID-induced inflammation and in IBD-pathogenesis. Interestingly, NLRP3 inhibition targeted the inflammatory response affecting macrophages leading to an increase in the number of CD206 + CD163 + M2 macrophages in the spleen and a reduced M1 macrophage gene expression profile in the colon, which is in line with a previous study in a model of valve stenosis and calcification. 45 No increase in M2 cells and a non-significant reduction in macrophages (CD11b + CD14 + ) were seen in the Caspase-8 inhibitor group, indicating potential differences in the regulation of macrophages by these proteins. Both inhibitors significantly reduced the presence of activated T-cells and associated cytokines such as IFNγ and IL-17α and reduced apoptotic and pyroptotic cell death, which probably contributed to the overall ameliorated phenotype presented by either of the inhibitor-treated groups.
Although no significant alterations in microbiota composition or individual SCFAs were detected in the inhibitor-treated groups, an increased abundance of Clostridium cluster XIVa was seen in both NLRP3 and Caspase-8 inhibitor groups. This is an SCFA producing bacteria, which can regulate Treg cells, and whose abundance is generally decreased in IBD. 46,47 A similar subtle effect on microbiota composition was reported in a model of experimental autoimmune encephalomyelitis (EAE) after NLRP3-inhibitor (MCC950) administration. 48 Although no increase in butyrate was noted in either of the inhibitor treatment groups, a higher induction of propionate and total SCFA was observed, potentially indicating the contribution of this genus to SCFA generation and reduced inflammation. The non-significant findings could also reflect the small group size comparisons. For this reason, future studies are warranted to further dissect the changes in microbial taxa following the use of the NLRP3 and Caspase-8 inhibitors.
In conclusion, our findings provide new insights into how NSAID and an opportunistic IBDassociated gut pathobiont can synergize to worsen IBD symptoms and inflammation. To the best of our knowledge, this is the first time that Caspase-8 has been reported as a potential regulator of AIEC and NSAID-induced colitis in vivo. Our data suggest that genetic factors alone are not enough to trigger pathogen-like activity by a pathobiont, and other factors such as environmental (exposure to NSAIDs) are needed to trigger a pathobiont's pathogenic potential. Further, we showed that targeting either Caspase-8 or NLRP3 could be a potential therapeutic strategy for IBD patients with NSAID-worsened inflammation. To date, there is no report in the literature that have examined or correlated the abundance of AIEC in patients with IBD and NSAIDs. We propose that future clinical IBD studies focusing on the role of NSAIDs should consider the status of the colonization of pathobionts such as AIEC as one of the factors influencing the inflammatory phenotype.

Limitation of study
One of the limitations of this study is the lack of information regarding the cell types in which activation of the NLRP3 inflammasome, Caspase-8, and biochemical markers of apoptosis and pyroptosis occurs. To mechanistically validate the findings from our model, we used human and murine epithelial cell lines (HT29, DLD1, CMT96) and primary cells including murine intestinal crypts and bone-marrow derived macrophages. However, none of these cell systems responded to in vitro activation with piroxicam, although previous studies have shown piroxicam-induced cell death in HCT116 and CaCo-2 cells. 49 With these limitations in mind, future studies are needed to further investigate the molecular and cellular mechanisms underpinning cross-regulation of the Caspase-8/NLRP3 axis. The generation of conditional knock-out mice for Caspase-8 and NLRP3 on an IL10 −/− background would be necessary to identify the cell specificity of Caspase-8/NLRP3 crosstalk and activation. Multiple staining for colocalization of ASC, Caspase-8, and NLRP3 on colon sections would also provide further information on cell-specific activation of these proteins and associated signaling complexes.

Reagents and resources
Providers and catalog numbers of reagents and resources are listed in Table 1 unless otherwise stated.

Bacterial strain and growth
Adherent-invasive E. coli HM605 stain was previously isolated from colonic biopsy of CD patients 32 and was provided by Dr David Clarke, School of Microbiology, UCC. For mouse oral gavage, E. coli HM605 was grown by culturing a single colony in 5 mL Luria Bertani (LB) Broth (Cat#L3022, Sigma-Aldrich) with 50 µg/mL ampicillin (Cat#A9518, Sigma) at 37°C overnight without shaking. The next day, the overnight culture was washed twice with PBS (Cat#P4417, Sigma) by centrifugation at 500 g for 10 min. After the second wash, the bacterial pellet was re-dispersed in PBS supplemented with 10% 1 M NaHCO 3 (Cat#S5761, Sigma) with the final OD 600 of 5. Each mouse was gavaged with 200 μL of bacterial culture with an OD 600 of 5.

Mice
IL10 −/− mice (B6.129P2-Il10tm1Cgn/J) were acquired from Charles River Laboratories, UK, and bred at the Bioscience Service Unit (BSU)-Annex at University College Cork. All animals were housed in individually ventilated cages (IVCs, OptiMICE, UK), with a controlled environment (20-22°C, 12 hours (hr) light:dark cycle) and given food and water ad libitum. All animal studies were designed with consideration for the three Rs (Replacement, Reduction, and Refinement) and were approved by the Animal Experimentation Ethics Committee (AEEC; applications #2018-028 and #2020/007) of University College Cork and by the Health Products Regulatory Authority (HPRA, project nrs AE19130/P101 and AE19130/P138).

Collection of samples
Colons were excised, opened longitudinally, and washed in PBS. Colon length was recorded, and colon was divided into two pieces with 3 cm of distal colon (weighed) and divided into two longitudinal pieces; one was snap frozen in liquid nitrogen for western blot analysis, and the other section was rolled, embedded in optimum cutting temperature (OCT, Cat#4583, Tissue-Tek™) compound and snap frozen in liquid nitrogen for histological analysis. For RNA isolation and RT-qPCR, a 0.5 cm piece of the most distal colon was stored in RNAlater (Cat#3335402001, Roche) for 24 hr at 4°C and snap frozen in liquid nitrogen. The spleen and MLNs were removed and processed for isolation of cells (see below). Feces and cecal content were collected and snap frozen in liquid nitrogen for 16S rDNA analysis. All collected samples were stored at −80°C until needed.

Bacterial counts
To enumerate the bacteria in the stool samples, fecal pellets were weighed and homogenized in PBS at a concentration of 0.1 mg/mL. Homogenized samples were plated onto LB agar with 50 µg/mL ampicillin. After culturing at 37°C overnight, bacterial counts were recorded.

Real time qPCR analysis
Total RNA from the colonic tissue was isolated using the Qiagen RNeasy kit (cat #74104 Qiagen) following the manufacturer's protocol and quantified using a NanoDrop Spectrophotometer (ND1000  Table 2.

Meso scale discovery (MSD) multiplex assay for cytokine protein quantification
Colonic tissue was homogenized as previously described. 52 Briefly, colon tissue was homogenized in 350 µL of lysis buffer (40 mL of PBS), 10% FCS (cat nr# F9665, Sigma) 2 Complete Protease Cocktail Inhibitor Tablets (Roche, Mannheim, Germany). Each sample was subject to three rounds of homogenization using a MagnaLyser (Roche) at 6,000 rpm for 15 seconds, placing the tubes on ice for 30 seconds between each round, followed by centrifugation at 10,000 g for 10 minutes at 4 °C. The supernatants/homogenates were aliquoted into fresh 1.5 mL tubes and stored at −80 °C until use. 25 µL of lysates per sample were used in MSD U-plex assay to quantify the cytokines IFNγ, IL-1β, IL-6, and mKC according to the manufacturer's instructions. Briefly, U-plex 10 assay plate was coated with linker-coupled biotinylated capture antibody overnight at 4°C. Next day, the plate was washed thrice with PBS containing 0.1% tween-20 (Cat# 1379 Sigma). After washing, samples and standards were added to the plate and incubated at room temperature (RT) for 1 hr with shaking, followed by three washes with PBS containing 0.1% tween-20 (PBST). Fifty microliters of secondary detection antibodies solution were added for 1 hr at RT with shaking following PBST washes. After the secondary incubation, the plate was washed thrice with PBST, followed by 150 µL of read buffer. The plate was read immediately after adding read buffer using MESO QuickPlex SQ 120. Data graphed as cytokine levels are expressed as pg cytokine/mg colonic tissue.

Western blot analysis
Western blot was performed as described with some modifications. 54  K-12042-D10, Advansta) and LAS-3000 Imager (Fujifilm) and processed with ImageJ software (without gel splicing and brightness/contrast adjustment). Antibodies used for western blotting are listed in Table 1.

Flow cytometric analysis
Spleens were removed, and single-cell suspensions were prepared as reported with some modifications. 55 Briefly, to isolate cells from the spleen, the tissue was pressed through a 100 μm cell strainer (Sarstedt, Germany) positioned on a 50 mL tube using the plunger of a 1 mL syringe and washing the strainer with 1X sterile PBS supplemented with 1% FCS. Samples were centrifuged for 5 min at 300 × g and red blood cells were lysed by 10 min incubation on 37℃ with 5 mL of 1 X RBC lysis buffer (eBioscience  Figure S3A-C). For T cell analysis, CD4 vs CD8 dot plots were obtained after gating cells with CD45 expression ( Figure S3A). For macrophage analysis, CD11b vs CD14 and CD11b vs F4/80 dot plots were obtained by gating on cells with CD45 expression ( Figure S3B). CD206 vs CD163 dot plots were obtained after gating on CD11b + F4/80 + cells ( Figure S3C).

DNA extraction, 16S rRNA gene sequencing, and gut microbiota analyses
DNA from fecal samples were extracted using a QIAamp Fast DNA Stool Mini Kit (Qiagen Cat# 51604) following the manufacturer's instructions. Amplicon sequencing of the 16S rDNA V3-V4 region of bacterial communities was performed as previously described, 56 utilizing 2x250bp pairedend Illumina MiSeq chemistry (Genewiz; Leipzig, Germany). Raw sequencing reads were processed using an in-house 16S rRNA processing pipeline, employing USEARCH (64 bit; v 8.1). Briefly, paired-end reads were merged and filtered using a < 0.5 expected error rate per nucleotide and total length. Reads were dereplicated, and singletons removed, following the trimming of the forward and reverse primers ("-stripleft 17" and "-stripright 21", respectively). Operational taxonomic units (OTUs) were clustered at 97% identity, and reference-based chimera removal was performed using UCHIME. OTUs were assigned taxonomic information by aligning reads to the RDP Gold database using the RDP Classifier (v 2.12). The 16S rRNA reads assigned taxonomic information was converted into a count matrix and imported into R Studio (v 3.6.1). The 16S data was analyzed using the metadata accompanying the relevant mouse trials and the RT-qPCR. The 16S rRNA OTU reads aligned per OTU were converted into relative abundances using the "funrar" package. 57 Dataframes and matrices were manipulated as necessary using the "reshape2" package. 58 Publication quality images were generated using the "ggplot2" and "ggpubr" packages. 59,60 Boxplots represent the standard Tukey representation, with boxes representing the 25th, 50th (median) and 75th interquartile range (IQR) percentiles and the whiskers encompassing values within 1.5× the IQR. The specific values of each boxplot are overplotted as opaque gray circular points using the ggplot "geom_jitter" function, whereas boxplot outliers are represented as solid square points.
The alpha-and beta-diversity of the samples were calculated using the R packages "vegan" and "phyloseq". 61,62 All alpha-diversity values presented use Simpson's index, while beta-diversity separation was performed using Canberra distances with PCoA ordination. A color palette for the composition bar plots was obtained through the "pals" R package. 63 The log 2 fold change of epithelial and inflammatory biomarker genes, and 16S rRNA taxa, was performed using the "DESeq2" package after scaling values to integers. 64 Correlations between microbial taxa and epithelial and inflammatory biomarker genes were performed using base R's "stats" package, 65 with plots generated using the "corrplot" package. 66 Individual images were manipulated into their final multipanel display using Inkscape (v 1.1.2).

Short chain fatty acids (SCFAs)
Analysis of SCFAs was performed as recently reported. 67 Briefly, cecal samples were weighed and diluted 1:10 (w/v) in sterile HPLC grade water. The SCFA-containing supernatant was filtered through 0.2 µm pore size cellulose acetate membrane (GyroDisc CA; Orange Scientific, Braine-l'Alleud, Belgium) and stored at −20°C until HPLC analysis. Quantification of SCFAs in cecal samples was carried out using an external calibration standard curve method. 67 Cecal SCFA concentrations were expressed as mean µmol per gram wet weight cecum using the following equation: Cecal SCFA (µmol/g) = [organic acid in cecal contents (mmol/mL) × Vd (ml) × 1000]/wet weight cecum (g), where Vd = total volume of dilution.

Statistical analysis
GraphPad Prism software was used to perform statistical analyses for all data sets except 16S amplicon data. All data were expressed as mean ± SEM Oneway ANOVA with Bonferroni test or Kruskal-Wallis with Dunn's multiple comparison test was used to test for significant differences between various groups. A p-value <0.05 was considered significant.
For analyzing the 16S amplicon data of the relevant mouse trials, the following statistical tests were performed. Wilcoxon tests were performed for nonparametric two-group comparisons, with the p-values presented above the relevant alpha-diversity box plots. Statistical significance in the beta-diversity of mice with respect to days post piroxicam, treatment group, or their interaction was assessed using the "adonis" function of the vegan R package, which performs a permutational multivariate analysis of variance (PERMANOVA) test. The variance (R 2 ) and p-values of PERMANOVA tests accompany the relevant PCoA plots. Statistically significant changes in microbial taxa with reference to mice treatment groups were calculated using Wilcoxon tests with Benjamini-Hochberg false discovery rate (fdr) correction. All correlations between microbial taxa and epithelial and inflammatory biomarkers were conducted using Spearman rank correlations.